freeshotgun sequencing library from miniprep BAC DNA

1.Inoculate 4 - 8 tubes of 3ml/tube LB with 15μg/ml chloramphenicol with BAC clone and grow oveernight. Miniprep with AutoGen 740 according to the protocol described in our Web page. DNA pellet were resuspended in 265 μl TE buffer pH 8.0 or ddH2 O.

2.NotI restriction digestion

3.265 μl miniprep DNA

4.30 μl of 10X New England BioLabs Buffer 3

5.5 μl of 10 units/μlNot 1 (New England BioLabs, #189)

6.incubate for 1hr at 37℃.

7.Purify insert DNA with PFGE

Add 60 μl of 6X DNA loading buffer into digestion mixture, load on 1% agarose - 0.5X TBE gel, run at 6V/cm, initial pulse 5 seconds and final pulse 15 seconds for 18 hrs with BioRAD CHEF-DR (r) II Pulse Field Gel Electrophoresis Apparatus. Load New England BioLabs Lambda Ladder PFG Marker ( #340) on the gel. Stain with ethidium bromind and evaluate.

8.Isolate inset DNA

Isolate DNA from agarose gel slice(s) depending on the insert size and with/without Not 1 cut site in the insert of individual BAC clone with gelase ( Gelase TM Agarose Gel-Digesting Properation, Epicentre Technologies, #09200) or QIAquick Gel Extraction Kit ( Qiagen, # 28704) , or GeneClean Kit for Purification of DNA from agarose gel ( Bio 101, #3105).

9.Sonication and end-repair

DNA pellet were resuspended in 100 μl ddH2 O and added 20 μl 10X MB buffer( 300 mM NaOAc pH5.0, 500 mM NaCl, 10mM ZnCl2 , 50% Glycerol), mixed in an Eppendorf tube. This mixture was sonicated for 2 - 3 seconds at 50% power in a Branson sonifier 250 on ice. For end repair, add 4 μl of 10 units/μlMung Bean Nuclease ( New England BIoLabs, #250 ), incubate for 25 minutes at 30℃. Purify DNA by phenol extraction/EtOH precipitation (-70℃ for 30 minuets).

10.Prepare size-selected fragments

Resuspend DNA pellet in 10 μl TE, pH 8.0, and add 2μl 6X DNA loading buffer, load on 1% agarose gel, side by side with Bohreenger Mannheim DNA Molecular Weight Marker VII 0.37 - 8.0 kbp (#1209 264). Run agarose gel for 2 hrs at 10 V/cm, stain with ethidium bromind and evaluate. Excise DNA in the range from 2 - 4kb from the agarose gel with a clean sharp scalpel, and isolate DNA from gel slices using QIAquick gel extraction kit. In the last step, use 30 μl of EB( elution buffer) to elute DNA from column and collect ~ 28 μl DNA.

11.Ligate

Ligate size selected DNA fragments into subvectors using Ready-To -Go TM pUC18 Smal1/BAP Ligase ( Phamarcia Biotech, #27-5266-01) Collected ~ 28 μl DNA in EB buffer were used as insert to be ligated directly. Add 20 μl DNA and 8 μl DNA plus 12 μl ddH2 O into two tubes of the Ready-To-Go respectively, and ligate at 15℃ overnight. Prior to electroporation, the ligation mixture must be cleaned according to the manufacture's protocol, and the EtOH should be remove completely from DNA pellet by airdry.

12.Electoporation

DNA pellet was resuspended in 4ul ddH2 O , chilled on ice, then added 20 - 40 μl of thawed ElectroMAX DH10B TM cell ( Life technologies, #18290-015). Mix DNA/Bacteria cell gently and incubate on ice for 2 minutes.

Transfer DNA/bacteria mixture into precooled 0.2 cm BioRed Gene Pulser(r) cuvette (#165-2086). Make sure that all of the mixture is positioned at the bottom of the cuvette between the electrodes.

Electroporate with the BioRad Gene Pulser with capacitance extender setting at 2.5 KV voltage, 200 ohm resistance, and a capacitance of 25 μF.

After the pulse, immediately add 1 ml of room temperature SOC medium and transfer the solution from cuvette into a culture tube, incubate at 37℃ for I hr with shaking at 225 rpm.

Plate 100 μl solution on an Ampicilin-selective LB agar plates containing X-Gal ( 40 μl of 20 mg/ml in dimethyformamide per plate) and IPTG ( 4 μl of 200 mg/ml in ddH2 O per plate) for blue/white screening. Grow plates overnight at 37℃. For each BAC clone, two ligation reactions and twice electroporations, finally get 20 transformation plates.

Pick up white colonies to generate shotgun sequencing library from total 20 plates for each BAC clone. Minimum number of colonies for 1X library is based on the equation ( Clarke & Carbon, 1976):

N = ln(1-p)/ln(1- f)

N, necessary number of colonies

p, the desired probability = 0.99

f, fractional proportion of the insert in a single colony

In our two pilot experiments: BAC A-589H1, containing 135 kb insert in which no Not 1 cut site, we pick up ~ 300 white colonies to grow and keep in three 96 well microtitres for 1X library. Each colony contains 100 μl overnight culture in LB with chloramphenicol plus 50 μl fresh LB in 50% Glycerol. BAC A-334D11, containing 180 kb insert in which no Not 1 cut site, we pick up ~ 400 white colonies to grow and keep in four 96 well microtitres for 1X library. From mentioned 20 plates for each BAC clone, we have 4 to 8 thousands white colonies for generation of 2X, 3X , even 4X shotgun sequencing library without E.coli DNA contamination.